A magnetic substance-containing structure, which is readily collectable by magnetic force, is promising for use mainly in biochemical fields as carriers of diagnostic agents, carriers for separation of bacteria or biological cells, carriers for separation and purification of nucleic acids or proteins, carriers for drug delivery, carriers for enzyme reaction, carriers for cell culture, and carriers for like uses. Further, the structure is promising as a carrier in drug-screening fields for selecting efficiently a target substance having an intended physiological activity or pharmacological activity from possible drug substances containing a biopolymer such as nucleic acids, peptides, proteins, and carbohydrates.
In such applications, in utilizing the magnetic substance-containing structure as a carrier, a means is necessary for immobilizing and holding the physiologically active substance on the surface of the magnetic substance-containing structure. Methods for the immobilization are disclosed in literature as below.
Japanese Patent Application Laid-Open No. H05-209884 discloses use of magnetic particles extracted from a magnetic bacteria as a carrier for immobilizing an antibody fragment on the surface of the magnetic particles. The magnetic particles derived from a magnetic bacterium are coated with a lipid film on the surface. On this lipid film, an antibody fragment is immobilized by utilizing N-succinimidyl 3-(2-pyridylthio)propionate.
Japanese Patent Application Laid-Open No. H05-080052 discloses immobilization of anti-rabbit IgG on ferrite particles prepared by coating of polymer particles mainly composed of polystyrene with Fe3O4 as the carrier modifying the particle surface by a—(CH2)3NHCO(CH2)3CONH(CH2)6NH2 group by use of a coupling agent.
Japanese Patent Application Laid-Open No. H07-063761 discloses a process for producing a fine particulate magnetic substance for immobilizing a physiologically active substance in which fine magnetic particles of average diameter of 0.3-1.0 μm are fixed onto resin particles of average diameter of 1.0-10 μm as the nuclei by a high-speed gas stream impact method; the fixed magnetic fine particles are surface-treated with a silane coupling agent; and a physiologically active substance is bonded thereon directly or through another functional group introduced thereto.
U.S. Pat. No. 5,776,360 discloses a method in which magnetite fine particles are surface treated for aminosilanation, and HCG (human chorionic gonadotropin) antibody is immobilized thereon with glutaraldehyde by utilizing the introduced amino group.
The aforementioned prior art techniques utilize a chemical covalent bond between the biosubstance and the magnetic substance for immobilizing a biosubstance on a magnetic substance contained in a carrier. Such a technique is liable to cause conversion or denaturation of the biosubstance depending on the covalent bond formation conditions (such as temperature, pH, and reagent). For example, the site exhibiting an inherent function of the biological material (e.g., molecular recognition site, and catalyst site) can be modified by introduction of a reagent to affect adversely the inherent function of the biosubstance. Otherwise, the covalent bond can be formed near the active site of the inherent function of the biosubstance to impair the inherent function exhibition.
As the results of the above adverse effects, the obtained biosubstance-holding carrier may not achieve the intended inherent function as the carrier of a medical diagnostic drug, the carrier for separation of bacteria or cells, the carrier for separation and purification of nucleic acids or proteins, the carrier for drug delivery, the carrier for enzyme reaction, the carrier for cell culture, or the carrier for drug screening.
The present invention provides a novel method for immobilizing a biosubstance like a protein on a carrier containing a magnetic substance, wherein the biosubstance is immobilized on the magnetic substance surface with the function kept active, and enabling immobilization of the intended biosubstance selectively onto the magnetic substance surface. The present invention provides also a magnetic substance-biosubstance complex structure, and a process for producing thereof.